Protein-based t-cell receptor knockdown

ABSTRACT

The invention relates to protein-based T-cell receptor knockdown, and its use in T-cell therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to Canadian PatentApplication No. 2,937,157 filed Jul. 25, 2016, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to protein-based T-cell receptor knockdown, andits use in T-cell therapies.

BACKGROUND TO THE INVENTION

Chimeric antigen receptor (CAR) T-cells graft the specificity of amonoclonal antibody (mAb) to a T-cell (Pule, M., Finney, H. & Lawson, A.Artificial T-cell receptors. Cytotherapy 5, 211-226 (2003)). CAR T-cellsare usually autologous—i.e. they are generated from the patient's ownlymphocytes. This is effective and simple but has a number oflimitations: (1) it may be difficult or impossible to generate a productfrom patient's own lymphocytes due to insufficient quantity or qualityof lymphocytes consequent to disease or chemotherapy; (2) there may beinsufficient time to generate an autologous CAR T-cell product due tothe tempo of the patient's illness; and (3) autologous productionrequires a bespoke product to be manufactured for each patient whichmakes manufacture costly.

An alternative approach is to generate “off-the-shelf” CAR T-cells fromhealthy donor lymphocytes. Cord blood lymphocytes are a particularlyconvenient source of donor lymphocytes for off-the-shelf CAR T-cellproduction. Using the off-the-shelf approach, production of the CART-cell product is independent of the patient. Furthermore, if themanufacturing process lends itself to economies of scale, theoff-the-shelf approach may advantageously reduce the cost of productionof the CAR T-cell product.

Given the wide variability of human leukocyte antigen (HLA) types, it isvery likely that any off-the-shelf CAR T-cell product will be completelyHLA-mismatched from the recipient. It is simply not feasible to have aHLA-matched, off-the-shelf CAR T-cell product ready for every recipientin need thereof. This HLA mismatch is associated with its own

technical challenges, in particular graft versus-host disease (GVHD). InGVHD, that native T-cell receptor (TCR) of T-cells in donated tissue(the “graft”) recognise antigens in the recipient (the “host”) asforeign. Thus, transplanted T-cells attack host cells and tissues,causing damage to the host organs. Acute or fulminant GVHD, whichnormally occurs within the first 100 days following transplant, isassociated with significant morbidity and mortality. Chronic GVHD, whichnormally occurs after 100 days following transplant, adverselyinfluences long-term survival.

GVHD typically occurs in the setting of allogeneic haematopoietic stemcell transplantation (HSCT), in which the donor and recipient are fullyor partially HLA-matched. In the off-the-shelf CAR T-cell approach, theCAR T-cell product and the recipient are completely HLA-mismatched. Whenthe donor and recipient are not matched, more severe type of GVHD knownas “transfusion-associated GVHD” (TA-GVHD) occurs.

In order to be of widespread utility, an off-the-shelf CAR T-cellproduct must cause, at most, a minimal amount of GVHD when administeredto a HLA-mismatched recipient. Current approaches to attenuating theability of HLA-mismatched CAR T-cells to cause GVHD involve editing thegenome of the CAR T-cells to disrupt native TCR expression, using zincfinger nucleases (ZFNs), transcription activator-like effector nucleases(TALENs), or the clustered regularly interspaced short palindromicrepeats (CRISPR)/Cas system. These genome editing methods can disrupt agene, entirely knocking out all of its output. However, several problemsare associated with these genome editing approaches. Firstly, the genesrequired for these approaches typically have to be delivered separatelyto CAR T-cells during their production, for instance by electroporationwith synthetic mRNA. Consequently, the resultant disruption or knockdownof the native TCR in the T-cell is not linked to CAR expression. Thismeans that sorting for CAR-expressing T-cells does not necessarily alsosort for T-cells expressing the genome editing genes required to disruptnative TCR expression. Likewise, sorting for expression of genomeediting genes does not necessarily sort for CAR-expression. Therefore,to obtain CAR T-cells suitable for use in an off-the-shelf product, itis necessary to perform two different sorting steps, one to select forCAR-expressing T-cells and one to select for T-cells expressing thegenome editing genes. Secondly, ZFNs, TALENS and CRISPR/Cas can allintroduce off-target gene disruptions and cause unwanted translocations.

An improved method of disrupting the expression of the native TCR in CART-cells is therefore required.

SUMMARY OF THE INVENTION

This invention relates to a novel mechanism by which to disrupt surfaceexpression of the native TCR in T-cells, such as CAR T-cells. Themechanism employed in the invention is not associated with thedisadvantages currently experienced with the existing, genome editingapproaches to disruption of the native T-cell receptor.

The inventors have surprising found that a molecule comprising a bindingportion comprising a binding domain which binds to one or morecomponents of TCR/CD3 complex, and a retention portion comprising aretention domain that retains the one or more components within theendoplasmic reticulum (ER) or Golgi apparatus may be used to disrupt theexpression of native TCR on the surface of a T-cell. By disrupting thesurface expression of native TCR, the capacity of the T-cell to causeGVHD following transfer to a HLA-mismatched patient is reduced orcompletely eliminated. Thus, the present invention relates the provisionof “universal” therapeutic T-cell, such as a CAR T-cell, for inclusionin an off-the-shelf immunotherapy product.

In the following description, references to expression of native TCRrefer to surface expression of functional TCR/CD3 complex.

When the T-cell is a CAR T-cell, the CAR and the molecule which disruptsexpression of the native TCR may be introduced to the T-cell together,using a nucleic acid construct that comprises a first nucleic acidsequence encoding the molecule and a second nucleic acid sequenceencoding the CAR. As set out in detail below, the first and secondnucleic acid sequences may be linked by a nucleic acid sequence thatallows the molecule and the CAR to be processed as separate, unfusedprotein products. In this way, expression of the molecule may be linkedto expression of the CAR. Thus, sorting for CAR-expressing T-cells alsosorts for T-cells expressing the molecule that disrupts native TCRexpression. Likewise, sorting for expression of the molecule thatdisrupts native TCR expression also sorts for CAR-expression. Therefore,only one sorting step is required to obtain CAR T-cells suitable for usein an off-the-shelf product.

Accordingly, the invention provides a molecule which disrupts theexpression of native TCR in a T-cell, which molecule comprises a bindingdomain which binds to one or more components of TCR/CD3 complex, and aretention portion comprising a retention domain that retains the one ormore components within the ER or Golgi apparatus.

The invention also provides:

-   a nucleic acid sequence encoding a molecule of the invention,    comprising a nucleic acid sequence encoding a binding portion    comprising a binding domain which binds to one or more components of    the TCR/CD3 complex, and a retention portion comprising a retention    domain that retains the one or more components within the ER or    Golgi apparatus;-   a nucleic acid construct comprising a first nucleic acid sequence of    the invention, and a second nucleic acid sequence which encodes a    chimeric antigen receptor (CAR);-   a nucleic acid construct comprising a first nucleic acid sequence of    the invention, and a second nucleic acid sequence which encodes a    suicide gene;-   a vector comprising the nucleic acid sequence of the invention or a    nucleic acid construct of the invention;-   a method for producing a T-cell expressing a molecule of the    invention, comprising: transfecting or transducing a T-cell with a    nucleic acid sequence of the invention or a nucleic acid construct    of the invention; and expressing the molecule in the T-cell;-   a T-cell comprising (a) a nucleic acid sequence of the invention or    a nucleic acid construct of the invention, or (b) a vector of the    invention;-   a method for reducing or completely eliminating expression of native    TCR in a T-cell, comprising (a) providing a nucleic acid sequence of    the invention or a nucleic acid construct of the invention, or a    vector encoding the nucleic acid sequence or nucleic acid    construct; (b) transfecting or transducing the T-cell with the    nucleic acid sequence, nucleic acid construct or vector; and (c)    expressing the molecule in the T-cell;-   a method for producing a T-cell having reduced or completely    eliminated expression of native TCR, comprising (a) providing a    T-cell; (b) transfecting or transducing the T-cell with a nucleic    acid sequence of the invention or a nucleic acid construct of the    invention, or a vector encoding the nucleic acid sequence or nucleic    acid construct; and (c) expressing the molecule in the T-cell;-   a method of reducing or preventing graft versus host disease (GVHD)    in a patient associated with the administration of one or more CAR    T-cells to the patient, comprising (a) transfecting or transducing    the one or more CAR T-cells with a nucleic acid sequence of the    invention or a nucleic acid construct of the invention, or a vector    encoding the nucleic acid sequence or nucleic acid construct;    and (b) administering the CAR T-cells to the patient;-   a method of reducing or preventing GVHD in a patient associated with    the transfusion of one or more CAR T-cells to the patient,    comprising (a) generating the one or more CAR T-cells by    transfecting or transducing one or more T-cells with a nucleic acid    construct comprising a first nucleic acid sequence of the invention    and a second nucleic acid sequence which encodes a chimeric antigen    receptor, or a vector encoding the nucleic acid construct; and (b)    administering the CAR T-cells to the patient;-   a method of reducing or preventing GVHD in a patient associated with    the transfusion of one or more CAR T-cells to the patient,    comprising (a) producing one or more T-cells expressing a molecule    of the invention by (i) providing one or more T-cells; (ii)    transfecting or transducing the one or more T-cells with a nucleic    acid sequence of the invention or a nucleic acid construct    comprising a first nucleic acid sequence of the invention and a    second nucleic acid sequence which encodes a suicide gene, or a    vector encoding the nucleic acid sequence or nucleic acid construct;    and (iii) expressing the molecule in the one or more T-cells; (b)    converting the one or more T-cells into one or more CAR T-cells by    transfecting or transducing the one or more T-cells with a construct    encoding a CAR and expressing the CAR; and (c) administering the CAR    T-cells to the patient;-   a nucleic acid sequence of the invention or a nucleic acid construct    comprising a first nucleic acid sequence of the invention and a    second nucleic acid sequence which encodes a suicide gene for use in    a method of reducing or preventing GVHD in a patient associated with    the administration of one or more CAR T-cells to the patient, the    method comprising (a) transfecting or transducing the one or more    CAR T-cells with the nucleic acid sequence or nucleic acid construct    and (b) administering the CAR T-cells to the patient;-   a nucleic acid construct comprising a first nucleic acid sequence of    the invention and a second nucleic acid sequence which encodes a    chimeric antigen receptor for use in a method of reducing or    preventing GVHD in a patient associated with the administration of    one or more CAR T-cells to the patient, the method comprising (a)    generating the one or more CAR T-cells by transfecting or    transducing one or more T-cells with the nucleic acid construct    and (b) administering the CAR T-cells to the patient;-   use of a nucleic acid sequence of the invention or a nucleic acid    construct comprising a first nucleic acid sequence of the invention    and a second nucleic acid sequence which encodes a suicide gene in    the manufacture of a medicament for the treatment of cancer or an    autoimmune condition, wherein the medicament comprises one or more    CAR T cells transfected or transduced with the nucleic acid sequence    or nucleic acid construct.-   use of a nucleic acid construct comprising a first nucleic acid    sequence of the invention and a second nucleic acid sequence which    encodes a chimeric antigen receptor in the manufacture of a    medicament for the treatment of cancer or an autoimmune condition,    wherein the medicament comprises one or more CAR T cells transfected    or transduced with the nucleic acid construct;-   a method of treating a disease in a patient in need thereof,    comprising administering to the patient a therapeutically effective    number of CAR T-cells expressing a molecule of the invention; and-   CAR T-cells expressing a molecule of the invention, for use in a    method of treating a disease in a patient in need thereof, the    method comprising administering to the patient a therapeutically    effective number of cells.

DESCRIPTION OF THE FIGURES

FIG. 1. Peripheral blood T-cells transduced with OKT3 and BMA031 basedsekdel constructs. Some knock-down of both transduced and non-transducedpopulations are seen. Anti-CTLA4 sekdel is used as a control.

FIG. 2. Annotated sequence for S-UCHT1-KDEL (SEQ ID NO:21).

FIGS. 3a and 3 b. (FIG. 3a ) structure of S-UCHT1-sekdel; (FIG. 3b )Mechanism of activity

FIGS. 4a and 4 b. (FIG. 4a ) TCR negative Jurkats and (FIG. 4b ) wtJurkats transduced with S-UCHT1-sekdel. Marker gene (eBFP2) is shown onthe x-axis. Transduced cells (i.e. eBFP2 cells) are TCR negative.

FIG. 5. Peripheral blood from two normal donors are transduced withS-UCHT1-sekdel.

FIG. 6. VH and VL sequences for UCHT1 and Jovi. 1. CDRs are shown inbold and highlighted. Shown are SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24and SEQ ID NO:25.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition, as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “amolecule” includes “molecules”, reference to “a T-cell” includes two ormore such T-cells, reference to “a component” includes two or more suchcomponents, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Molecule of the Invention

The present invention provides a molecule which disrupts the expressionof native TCR in a T-cell, which molecule comprises a binding domainwhich binds to one or more components of the TCR/CD3 complex, and aretention portion comprising a retention domain that retains the one ormore components within the ER or Golgi apparatus.

Disruption of the expression of the native TCR refers to changing theamount of expression of native TCR on the surface of the T-cell.Preferably, the expression of native TCR is reduced or completelyeliminated. A T-cell having reduced expression of native TCR has areduced amount of native TCR on its surface. A T-cell with completelyeliminated expression of native TCR has no native TCR on its surface.

Methods for determining surface expression of native TCR are known inthe art. For instance, T-cells surface-stained (i.e. stained without apermeablisation step) with an antibody or other molecule that binds tothe TCR may be analysed by flow cytometry or fluorescence microscopy.Using flow cytometry, reduction in the mean fluorescence intensity (MFI)of TCR surface-staining in study T-cells compared to control T-cellsindicates a reduction in surface expression of native TCR. For instance,the MFI (and thus surface expression) may be reduced by up to 100%, suchas up to 99%, up to 98%, up to 95%, up to 90%, up to 85%, up to 80%, upto 75%, up to 70%, up to 60%, up to 50%, up to 40%, or up to 25%, instudy T-cells compared to control T-cells. The absence of native TCRexpression is indicated by the absence of surface-staining for thenative TCR (i.e. by an MFI equivalent or similar to that of a negativecontrol sample).

The TCR/CD3 complex, otherwise known as the T-cell receptor complex, isa multimeric complex on the T-cell surface whose activation leads to theactivation of the T-cell. The complex comprises (i) TCR, (ii) CD3 T-cellco-receptor. As set out below, the TCR comprises alpha (α) and beta (β)chains. The CD3 T-cell co-receptor comprises a CD3-gamma (CD3γ) chain, aCD3-delta (CD38) chain, two CD3-epsilon (CD3s) chains and two zeta-chain(ζ-chain) accessory molecules.

TCRs allow for the antigen-specific activation of T-cells. Every T-cellexpresses clonal TCRs which recognize specific peptide/MHC complexduring physical contact between T-cell and antigen-presenting cell-APC(via MHC class II) or any other cell type (via MHC class I). The TCR isa disulfide-linked membrane-anchored heterodimeric protein normallyconsisting of the highly variable alpha (α) and beta (β) chains. Eachchain of the TCR comprises two extracellular domains: a variable (V)region and a constant (C) region, both of immunoglobulin superfamily(IgSF) domain forming antiparallel β-sheets. The constant region isproximal to the cell membrane, followed by a transmembrane region and ashort cytoplasmic tail, while the variable region binds to thepeptide/MHC complex. The variable domain of the TCR α-chain and the TCRP-chain each have three hypervariable or complementarity determiningregions (CDRs), that contribute to the TCR's specificity for aparticular peptide/MHC complex. The variable region of the β-chain alsohas an additional area of hypervariability (HV4) that does not normallycontact antigen.

CD3 is required for the antigen-specific activation of T-cells. Inparticular, CD3 links antigen recognition by the TCR with intracellularsignalling events downstream of the TCR T3 zeta-chain. CD3 is a proteincomplex comprising six distinct chains. In mammals, CD3 comprises aCD3-gamma (CD3γ) chain, a CD3-delta (CD3δ) chain, two CD3-epsilon (CD3ε)chains and two zeta-chain (ζ-chain) accessory molecules. The CD3-gamma,CD3-delta and CD3-epsilon chains are highly related cell-surfaceproteins of the immunoglobulin superfamily comprising a singleextracellular immunoglobulin domain. The transmembrane region of the CD3chains is negatively charge, allowing the chains to associated with TCRchains, which are positively charged. The zeta-chain (also known asT-cell surface glycoprotein CD3 zeta-chain or CD247) plays an importantrole in coupling antigen recognition to several intracellularsignal-transduction pathways. Low expression of the zeta-chain resultsin an impaired immune response.

Accordingly, TCR/CD3 normally comprises several components: TCR α-chain,TCR β-chain, CD3-gamma chain, CD3-delta chain, two CD3-epsilon chains,and two CD3-zeta-chains. The molecule of the invention binds to one ormore of these components. For example, the molecule of the invention maybind to two or more, three or more, four or more, five or more, or sixor more of these components. When the molecule binds to two or more of(i) TCR α-chain, (ii) TCR β-chain, (iii) CD3-gamma chain, (iv) CD3-deltachain, (v) CD3-epsilon chain, and (v) zeta-chain, it may bind thesecomponents in any combination, i.e. (i,ii); (i,iii); (i,iv); (i,v);(i,vi); (ii,iii); (ii,iv); (ii,v); (ii,vi); (iii,iv); (iii,v); (iii,vi);(iv,v); (iv,vi); (v,vi); (i,ii,iii); (i,ii,iv); (i,ii,v); (i,ii,vi);(i,iii,iv); (i,iii,v); (i,iii,vi); (i,iv,v); (i,iv,vi); (i,v,vi);(ii,iii,iv); (ii,iii,v); (ii,iii,vi); (ii,iv,v); (ii,iv,vi); (ii,v,vi);(iii,iv,v); (iii,iv,vi); (iii,v,vi); (iv,v,vi); (i,ii,iii,iv);(i,ii,iii,v); (i,ii,iii,vi); (i,ii,iv,v); (i,ii,iv,vi); (i,ii,v,vi);(i,iii,iv,v); (i,iii,iv,vi); (i,iii,v,vi); (i,iv,v,vi); (ii,iii,iv,v);(ii,iii,iv,vi); (ii,iii,v,vi); (ii,iv,v,vi); (iii,iv,v,vi);(i,ii,iii,iv,v); (i,ii,iii,iv,vi); (i,ii,iii,v,vi); (i,ii,iv,v,vi);(i,iii,iv,v,vi); (ii,iii,iv,v,vi); or (i,ii,iii,iv,v,vi). The moleculemay also bind to two or more (such as three or more, four or more orfive or more) of the same component, such as two or more TCR α-chains,two or more TCR β-chains, two or more CD3-gamma chains, two or moreCD3-delta chains, two or more CD3-epsilon chains, or two or morezeta-chains.

The one or more components to which binding occurs may be assembled toform the TCR/CD3 complex. That is, the one or more components may bepart of a complex comprising TCR α-chain, TCR β-chain, CD3-gamma chain,CD3-delta chain, two CD3-epsilon chains, and two zeta-chains.

Preferably, the one or more components to which bind occurs are notassembled to form the TCR/CD3 complex. That is, the one or morecomponents are preferably not part of a complex comprising TCR α-chain,TCR β-chain, CD3-gamma chain, CD3-delta chain, two CD3-epsilon chains,and two zeta-chains. In other words, binding may occur to a singlecomponent that is not associated with any of the other components.Binding may occur to one or more components that are associated witheach other but which do not from the full TCR/CD3 complex. Thus, bindingmay occur to a nascent, incomplete form of the TCR/CD3 complex. In thiscase, the one or more components may not form any complex. The one ormore components may form a complex other than the TCR/CD3 complex. Theone or more components may form a complex that comprises some but notall of the other components of the TCR/CD3 complex, such as a complexcomprising (i,ii); (i,iii); (i,iv); (i,v); (i,vi); (ii,iii); (ii,iv);(ii,v); (ii,vi); (iii,iv); (iii,v); (iii,vi); (iv,v); (iv,vi); (v,vi);(i,ii,iii); (i,ii,iv); (i,ii,v); (i,ii,vi); (i,iii,iv); (i,iii,v);(i,iii,vi); (i,iv,v); (i,iv,vi); (i,v,vi); (ii,iii,iv); (ii,iii,v);(ii,iii,vi); (ii,iv,v); (ii,iv,vi); (ii,v,vi); (iii,iv,v); (iii,iv,vi);(iii,v,vi); (iv,v,vi); (i,ii,iii,iv); (i,ii,iii,v); (i,ii,iii,vi);(i,ii,iv,v); (i,ii,iv,vi); (i,ii,v,vi); (i,iii,iv,v); (i,iii,iv,vi);(i,iii,v,vi); (i,iv,v,vi); (ii,iii,iv,v); (ii,iii,iv,vi); (ii,iii,v,vi);(ii,iv,v,vi); (iii,iv,v,vi); (i,ii,iii,iv,v); (i,ii,iii,iv,vi);(i,ii,iii,v,vi); (i,ii,iv,v,vi); (i,iii,iv,v,vi); or (ii,iii,iv,v,vi).

T-cell receptor assembly is reviewed by Call et al., (Molecularmechanisms for the assembly of the T-cell receptor-CD3 complex. Mol.Immunol. 40, 1295-1305 (2004)). Assembly results in alignment of polarresidues in the transmembrane domain. Each assembly step thus results inthe formation of a three-helix interface in the membrane that involvesone basic and two acidic transmembrane residues, and this arrangementeffectively shields these ionizable residues at protein-proteininterfaces from the lipid. Since proteins whose transmembrane domainshave exposed ionizable residues are not stably integrated into the lipidbilayer, assembly based on shielding of ionizable residues permits fullequilibration of the receptor into the lipid bilayer and preventsdegradation. Assembly, export of intact receptor complexes is preciselyregulated with degradation of unassembled components. The CD3/TCRcomplex hence is quite vulnerable to strategies which perturb itsassembly, such as binding by the binding portion of the molecule andretention in the ER or Golgi apparatus by the retention portion of themolecule.

The binding domain may be any domain that is capable of binding to oneor more of the components of TCR/CD3 complex. Thus, the binding domainmay be any domain that is capable of binding to one or more of (i) TCRα-chain, (ii) TCR β-chain, (iii) CD3-gamma chain, (iv) CD3-delta chain,(v) CD3-epsilon chain, and (v) zeta-chain. For instance, the bindingdomain may bind to (i); (ii); (iii); (iv); (v); (vi); (i,ii); (i,iii);(i,iv); (i,v); (i,vi); (ii,iii); (ii,iv); (ii,v); (ii,vi); (iii,iv);(iii,v); (iii,vi); (iv,v); (iv,vi); (v,vi); (i,ii,iii); (i,ii,iv);(i,ii,v); (i,ii,vi); (i,iii,iv); (i,iii,v); (i,iii,vi); (i,iv,v);(i,iv,vi); (i,v,vi); (ii,iii,iv); (ii,iii,v); (ii,iii,vi); (ii,iv,v);(ii,iv,vi); (ii,v,vi); (iii,iv,v); (iii,iv,vi); (iii,v,vi); (iv,v,vi);(i,ii,iii,iv); (i,ii,iii,v); (i,ii,iii,vi); (i,ii,iv,v); (i,ii,iv,vi);(i,ii,v,vi); (i,iii,iv,v); (i,iii,iv,vi); (i,iii,v,vi); (i,iv,v,vi);(ii,iii,iv,v); (ii,iii,iv,vi); (ii,iii,v,vi); (ii,iv,v,vi);(iii,iv,v,vi); (i,ii,iii,iv,v); (i,ii,iii,iv,vi); (i,ii,iii,v,vi);(i,ii,iv,v,vi); (i,iii,iv,v,vi); (ii,iii,iv,v,vi); or(i,ii,iii,iv,v,vi). Assays suitable for determining the ability of abinding domain to bind to one or more of the components of TCR/CD3complex are well known in the art, such as Western blotting orenzyme-linked immunosorbent assay (ELISA).

The binding domain preferably binds to one or more of the components ofthe TCR/CD3 complex, wherein the one or more components are notassembled to form the TCR/CD3 complex. That is, the binding domainpreferably binds to the one or more components when they are not part ofa complex comprising TCR α-chain, TCR β-chain, CD3-gamma chain,CD3-delta chain, two CD3-epsilon chains, and two zeta-chains.

Preferably, the binding domain may be used to stain intracellularly forone or more of the components of the TCR/CD3 complex. In other words,the binding domain is preferably a molecule that can be used todetermine the intracellular presence of one or more components of theTCR/CD3 complex by, for instance, immunofluorescent methods (such asflow cytometry or fluorescence microscopy) or immunohistochemistry. Theability of the binding domain to stain intracellularly for one or moreof the components of the TCR/CD3 complex may correlate with the abilityof the binding domain to recognise assembling TCR/CD3 complex in anascent stage before access to the ER or Golgi apparatus has passed.

The binding domain may be a superantigen (SAg), a TCR agonist, anantibody, a monoclonal antibody, a fragment antigen-binding (Fab)fragment, a F(ab′)₂ fragment, a single chain variable fragment (scFv), ascFv-Fc, or a single domain antibody. The binding domain is a preferablya scFv, a scFv-Fc, a single domain antibody or a monoclonal antibody.The single domain antibody is preferably a camelid antibody, anartificial V_(H)H fragment or an IgNAR. The binding domain is mostpreferably a scFv.

The binding domain preferably binds to CD3-epsilon. More preferably, thebinding domain is a UCHT1 antibody, or is derived from a UCHT1 antibody.Thus, the binding domain may be produced by or derived from a product ofa UCHT1 hybridoma.

The binding domain may be a UCHT1-derived scFv, scFv-Fc, single domainantibody or monoclonal antibody. For instance, the binding domain maycomprise the heavy chain complementarity determining region (CDR)1, CDR2and CDR3 sequences of SEQ ID NOs: 1, 2 and 3 respectively. The bindingdomain may comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQID NOs: 4, 5 and 6 respectively. The binding domain may comprise theheavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3respectively and the light chain CDR1, CDR2 and CDR3 sequences of SEQ IDNOs: 4, 5 and 6 respectively. The binding domain may comprise a heavychain variable region (HCVR) comprising the heavy chain CDR1, CDR2 andCDR3 sequences of SEQ ID NOs: 1, 2 and 3 respectively. The bindingdomain may comprise a light chain variable region (LCVR) comprising thelight chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6respectively. The binding domain may comprise a HCVR comprising theheavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3respectively and a LCVR comprising the light chain CDR1, CDR2 and CDR3sequences of SEQ ID NOs: 4, 5 and 6 respectively. The binding domain maycomprise the HCVR sequence of SEQ ID NO:7. The binding domain maycomprise the LCVR sequence of SEQ ID NO:8. The binding domain maycomprise the HCVR sequence of SEQ ID NO:7 and the LCVR sequence of SEQID NO:8. The binding domain may comprise three heavy chain CDRs (HCDR1,HCDR2 and HCDR3) contained within the HCVR sequence of SEQ ID NO: 7. Thebinding domain may comprise three light chain CDRs (LCDR1, LCDR2 andLCDR3) contained within the LCVR sequence of SEQ ID NO: 8. The bindingdomain may comprise three heavy chain CDRs (HCDR1, HCDR2 and HCDR3)contained within the HCVR sequence of SEQ ID NO: 7 and three light chainCDRs (LCDR1, LCDR2 and LCDR3) contained within the LCVR sequence of SEQID NO: 8. The binding domain may comprise a heavy chain comprising theHCVR sequence of SEQ ID NO: 7. The binding domain may comprise a lightchain comprising the LCVR sequence of SEQ ID NO: 8. The binding domainmay comprise a heavy chain comprising the HCVR sequence of SEQ ID NO: 7and a light chain comprising the LCVR sequence of SEQ ID NO: 8.

The binding domain may further comprise one or more of the constantdomains that are normally included in an UCHT1 antibody (i.e. anantibody produced by an UCHT1 hybridoma). The binding domain maycomprise one or more constant domains other than those that are includedin an UCHT1 antibody. The binding domain may comprise a combination ofone or more of the constant domains that are normally included in anUCHT1 antibody, and one or more constant domains other than those thatare included in an UCHT1 antibody.

The binding domain preferably binds to TCR beta chain. More preferably,the binding domain is a Jovi.1 antibody, or is derived from a Jovi.1antibody. Thus, the binding domain may be produced by or derived from aproduct of a Jovi.1 hybridoma.

The binding domain may be a Jovi.1-derived derived scFv, scFv-Fc, singledomain antibody or monoclonal antibody. For instance, the binding domainmay comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ IDNOs: 9, 10 and 11 respectively. The binding domain may comprise thelight chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 12, 13 and 14respectively. The binding domain may comprise the heavy chain CDR1, CDR2and CDR3 sequences of SEQ ID NOs: 9, 10 and 11 respectively and thelight chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 12, 13 and 14respectively. The binding domain may comprise a HCVR comprising theheavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 9, 10 and 11respectively. The binding domain may comprise a LCVR comprising thelight chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 12, 13 and 14respectively. The binding domain may comprise a HCVR comprising theheavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 9, 10 and 11respectively and a LCVR comprising the light chain CDR1, CDR2 and CDR3sequences of SEQ ID NOs: 12, 13 and 14 respectively. The binding domainmay comprise the HCVR sequence of SEQ ID NO: 15. The binding domain maycomprise the LCVR sequence of SEQ ID NO: 16. The binding domain maycomprise the HCVR sequence of SEQ ID NO: 15 and the LCVR sequence of SEQID NO: 16. The binding domain may comprise three heavy chain CDRs(HCDR1, HCDR2 and HCDR3) contained within the HCVR sequence of SEQ IDNO: 15. The binding domain may comprise three light chain CDRs (LCDR1,LCDR2 and LCDR3) contained within the LCVR sequence of SEQ ID NO: 16.The binding domain may comprise three heavy chain CDRs (HCDR1, HCDR2 andHCDR3) contained within the HCVR sequence of SEQ ID NO: 15 and threelight chain CDRs (LCDR1, LCDR2 and LCDR3) contained within the LCVRsequence of SEQ ID NO: 16. The binding domain may comprise a heavy chaincomprising the HCVR sequence of SEQ ID NO: 15. The binding domain maycomprise a light chain comprising the LCVR sequence of SEQ ID NO: 16.The binding domain may comprise a heavy chain comprising the HCVRsequence of SEQ ID NO: 15 and a light chain comprising the LCVR sequenceof SEQ ID NO: 16.

The binding domain may further comprise one or more of the constantdomains that are normally included in an Jovi.1 antibody (i.e. anantibody produced by a Jovi.1 hybridoma). The binding domain maycomprise one or more constant domains other than those that are includedin a Jovi.1 antibody. The binding domain may comprise a combination ofone or more of the constant domains that are normally included in anJovi.1 antibody, and one or more constant domains other than those thatare included in an Jovi.1 antibody.

The molecule may comprise two or more, such as three or more, four ormore, five or more binding domains. In this case, two or more of thebinding domains may be the same. Two or more of the binding domains maybe different.

The retention domain may be any domain that retains the one or morecomponents of the TCR/CD3 complex within the ER or Golgi apparatus. Theretention domain may be a target peptide. A target peptide is a shortpeptide chain of 3 to 70 amino acids that directs the transport of aprotein to a specific region of the cell, such as the ER or the Golgiapparatus, and/or retains the protein in the specific region. A varietyof target peptides are known in the art.

The retention domain is preferably a KDEL sequence, a KKXX motif, aKXKXX motif, a tail of adenoviral E19 protein having sequenceKYKSRRSFIDEKKMP, or a fragment of HLA invariant chain having sequenceMHRRRSRSCR. The retention domain is preferably C-terminal to the bindingdomain. The retention domain may be N-terminal to the binding domain.

The retention domain is preferably a KDEL (Lys-Asp-Glu-Leu)(SEQ ID NO:17) sequence. KDEL is a target peptide sequence in the amino acidstructure of a protein which prevents the protein from being secretedfrom the ER. A protein having a KDEL sequence will be retrieved from theGolgi apparatus by retrograde transport to the ER lumen. The KDELsequence may also target proteins from other locations (such as thecytoplasm) to the ER. Proteins can only leave the ER after the KDELsequence has been cleaved off. Thus, the protein resident in the ER willremain in the ER as long as it contains a KDEL sequence.

The retention domain may be a KKXX (Lys-Lys-xxx-xxx) motif. KKXX is atarget peptide motif that is generally located in the C terminus of theamino acid structure of a protein. KKXX is responsible for retrieval ofER membrane proteins from the cis end of the Golgi apparatus byretrograde transport, via interaction with the coat protein (COPI)complex.

The retention domain may be a C-terminal cytoplasmic tail of a known ERprotein, such as adenoviral E19 protein. For instance, the bindingdomain may be a tail of adenoviral E19 protein having sequenceKYKSRRSFIDEKKMP (SEQ ID NO: 18). Alternatively, the binding domain maybe an N-terminal fragment of the invariant chain of HLA, such as afragment having sequence MHRRRSRSCR (SEQ ID NO: 19).

Other suitable binding domains are known in the art. For instance,LocSigDB (http://genome.unmc.edu/LocSigDB/) is a database ofexperimental protein localization signals for eight distinct subcellularlocations (Negi et al., LocSigDB: a database of protein localizationsignals, Database (Oxford), 2015, 1-7). Furthermore, known methods maybe used to identify further binding domains that retains the one or morecomponents of the TCR/CD3 complex within the ER or Golgi apparatus (see,for example, Bejarano and Gonzalez, Motif Trap: a rapid method to clonemotifs that can target proteins to defined subcellular localisations,Journal of Cell Science, 112, 4207-4211 (1999)).

The molecule may comprise two or more, such as three or more, four ormore, five or more retention domains. In this case, two or more of theretention domains may be the same. Two or more of the retention domainsmay be different.

Nucleic Acid Sequence of the Invention

The invention also provides a nucleic acid sequence encoding a moleculeof the invention, comprising a nucleic acid sequence encoding a bindingportion comprising a binding domain which binds to one or morecomponents of TCR/CD3 complex, and a retention portion comprising aretention domain that retains the one or more components within the ERor Golgi apparatus.

The nucleic acid sequence may comprise DNA and/or RNA. The nucleic acidsequence may be double stranded or single stranded. For instance, thenucleic acid sequence may comprise dsDNA and/or ssDNA. The nucleic acidsequence may comprise dsRNA and/or ssRNA.

Any of the one or more components, the binding domain or the retentiondomain may be as discussed above with reference to the molecule of theinvention.

Nucleic Acid Constructs of the Invention

The invention further provides a nucleic acid construct comprising afirst nucleic acid sequence of the invention, and a second nucleic acidsequence which encodes a CAR.

The nucleic acid construct may comprise DNA and/or RNA. The nucleic acidconstruct may be double stranded or single stranded. For instance, thenucleic acid construct may comprise dsDNA or ssDNA. The nucleic acidconstruct may comprise dsRNA and/or ssRNA. Similarly, the first and/orsecond nucleic acid sequence may comprise DNA and/or RNA. The firstand/or second nucleic acid sequence may be double stranded or singlestranded. The first and/or second nucleic acid sequence may comprisedsDNA and/or ssDNA. The first and/or second nucleic acid may comprisedsRNA and/or ssRNA.

The construct may comprise two or more, such as three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more or twenty or more of each type of nucleic acidsequence.

CARs may be used to re-direct the antigen specificity of a T-cell. CARstypically comprise the heavy (V_(H)) and light (V_(L)) variablefragments from an antibody joined by a short linker to form a scFv. ThescFv is attached to a spacer region that allows it to protrude from thecell surface. The spacer region may be the CD8 stalk. A transmembranedomain then links the extracellular domain (ectodomain) to one or moreintracellular activation domains. These may be immunoreceptortyrosine-based inhibition motifs (ITIMs) or immunoreceptortyrosine-based activation motif (ITAMs) depending on the intracellularsignaling pathway to be activated or inhibited. Commonly usedintracellular activation domains include the CD3-zeta, CD28, OX40 and41BB intracellular signaling domains. Thus, ligand binding to the CARmay lead to activation of the intracellular signaling cascade and T-cellactivation by initiating phosphorylation of ITAMs. Conversely, ligandbinding to the CAR may initiate phosphorylation of ITIMs, givinginhibition of the intracellular signaling cascade and T-cell inhibition.

In this way, CARs are able to activate T-cells in response to targetcell surface antigens without the need for major histocompatibilitycomplex (MHC) recognition. Therefore, CARs can easily modify mostsubsets of T-cells to have increased persistence and improveddevelopment of memory. Furthermore, CARs are not restricted torecognition of protein-derived peptides. CARs may recognise other typesof cell surface molecule on a target cell, including non-proteinstructures such as gangliosides and carbohydrate antigens.

Accordingly, the CAR encoded by the second nucleic acid sequence may bespecific for an extracellular antigen or a cell surface antigen. Theantigen may be a peptide antigen or a non-peptide antigen. Thenon-peptide antigen may be a carbohydrate antigen, a ganglioside, or aglycolipid. The CAR may be specific for a tumour antigen, a viralantigen, a bacterial antigen, a fungal antigen, a protozoal antigen, ahost antigen or a cytokine. The CAR may be specific for one or moreantigens associated with acute myeloid leukemia (AML), such as one ormore of CD33, CD123 and CLL1.

The provision of a single nucleic acid construct comprising a firstnucleic acid sequence of the invention, and a second nucleic acidsequence which encodes a CAR may be advantageous over the provision oftwo separate amino acid sequences each encoding one of these components.The inclusion of the sequence encoding the molecule of the invention andthe sequence encoding a CAR in the same construct allows reduction orabrogation of native TCR expression by the molecule of the invention tobe linked with CAR receptor. T-cells transfected or transformed with theconstruct will express both the molecule of the invention, and a CAR.Therefore, T-cells transfected or transformed with the construct willexpress the encoded CAR, but will not express (or will express a reducedamount) of native TCR. Therefore, sorting (by e.g. magnetic-activatedcell sorting (MACS)) for T-cells that do not express native TCR willalso sort for CAR T-cells. Conversely, sorting for CAR T-cells will alsosort for cells that do not express native TCR, or express a reducedamount of native TCR.

The nucleic acid construct of the invention may further comprise a thirdnucleic acid sequence which encodes a suicide gene. The construct maycomprise two or more, such as three or more, four or more, or five ormore sequences encoding a suicide gene. Suicide genes are discussed indetail below.

The invention also provides a nucleic acid construct comprising a firstnucleic acid sequence of the invention, and a second nucleic acidsequence which encodes a suicide gene.

The nucleic acid construct may comprise DNA and/or RNA. The nucleic acidconstruct may be double stranded or single stranded. For instance, thenucleic acid construct may comprise dsDNA or ssDNA. The nucleic acidconstruct may comprise dsRNA and/or ssRNA. Similarly, the first and/orsecond nucleic acid sequence may comprise DNA and/or RNA. The firstand/or second nucleic acid sequence may be double stranded or singlestranded. The first and/or second nucleic acid sequence may comprisedsDNA and/or ssDNA. The first and/or second nucleic acid may comprisedsRNA and/or ssRNA.

The construct may comprise two or more, such as three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, ten or more or twenty or more of each type of nucleic acidsequence.

A suicide gene is a gene that causes a cell to kill itself throughapoptosis. Activation of these genes can be due to many processes, butthe main cellular “switch” to induce apoptosis is the p53 protein. Asdiscussed above, CAR expression can be used to redirect a T-cell'sresponse towards an antigen of interest, for example a tumour antigen orviral antigen. Therefore, CAR T-cells can be used therapeutically toenhance T-cell responses against e.g. tumour cells or virus-infectedcells. However, CAR T-cells are also capable of eliciting damagingside-effects in the host. For instance, administration of CAR T-cellsmay lead to cytokine release syndrome, neurologic toxicity, “ontarget/off tumour” recognition, anaphylaxis, clonal expansion secondaryto insertional oncogenesis, off-target antigen recognition, and, asdiscussed above, GVHD (Bonifant et al.3 Toxicity and management in CART-cell therapy, Molecular Therapy—Oncolytics, 3, Article number: 16011(2016)). The incorporation of a suicide gene to a CAR T-cell provides amechanism by which such CAR T-cell induced toxicities may be reduced oreliminated.

The suicide gene encoded by the construct of the invention may thereforact as a safety switch to limit toxicities induced by CAR T-celladministration. Any suitable suicide gene may be used. Suicide genes arewell-know in the art. The suicide gene is preferably RQR8, iCasp9 orthymidine kinase. The suicide gene preferably allows cells expressingthe suicide gene to be selectively deleted in response to administrationof the substance. For example, RQR8 facilitates selective deletion ofcells expressing this gene upon exposure to rituximab. Similarly, iCasp9facilitates selective deletion of cells expressing this gene uponexposure to AP1903. Thymidine kinase allows cells expressing this geneto be killed using ganciclovir.

As the sequence encoding the suicide gene is comprised in the samenucleic acid construct as the sequence encoding the molecule of theinvention, the reduction or abrogation of native TCR expression by themolecule of the invention will be linked in cells transformed ortransfected with the construct with expression of the suicide gene.T-cells transfected or transformed with the construct will express boththe molecule of the invention, and the suicide gene. Therefore, T-cellstransfected or transformed with the construct will possess the encodedsuicide gene, but will not express (or will express a reduced amount) ofnative TCR. Therefore, sorting (by e.g. FACS) for T-cells that do notexpress native TCR will also sort for T-cells possessing the suicidegene. Conversely, sorting for T-cells possessing the suicide gene willalso sort for cells that do not express native TCR, or express a reducedamount of native TCR.

In some situations, the construct comprises (a) a nucleic acid sequenceencoding the molecule of the invention, (b) a nucleic acid sequenceencoding a CAR, and (c) a nucleic acid sequence encoding a suicide gene.In T-cells transfected or transformed with such a construct, expressionof the molecule, the CAR and the suicide gene will be coupled.Therefore, T-cells transfected or transformed with the construct willpossess the encoded CAR and suicide gene, but will not express (or willexpress a reduced amount) of native TCR. Therefore, sorting (by e.g.FACS) for T-cells that do not express native TCR will also sort forT-cells possessing the CAR and the suicide gene. Sorting for T-cellspossessing the CAR will also sort for cells that possess the suicidegene but do not express (or express a reduced amount of) native TCR.Likewise, sorting for T-cells possessing the suicide gene will also sortfor cells that express the CAR but do not express (or express a reducedamount of) native TCR.

As set out above, the construct of the invention comprises two or morenucleic acid sequences, each encoding a different protein. Thus, theconstruct of the invention encodes multiple different proteins. To allowthe multiple proteins to be encoded as polyproteins that dissociate intoseparate protein products following translation, one or more of thenucleic acid sequences comprised in the construct may be linked by anucleic acid sequence encoding a self-processing peptide, such as afoot-and-mouth disease 2A-like peptide (2A peptide for short).Foot-and-mouth disease 2A-like peptide is described in detail inDonnelly et al., J Gen Virol. 2001 May; 82 (Pt 5): 1027-1041. Peptidebond formation between glycine and proline residues within the 2Apeptide is highly inefficient and, as a result, the proteins encoded bytwo or more nucleic acid sequences linked by a sequence encoding a 2Apeptide will be stoichiometrically processed as multiple unfused proteinproducts.

Vector of the Invention

The invention also provides a vector comprising the nucleic acidsequence of the invention or a nucleic acid construct of the invention.

The vector may be a viral vector. Preferably, the viral vector is alentivirus, a retrovirus, an adenovirus, an adeno-associated virus(AAV), a vaccinia virus or a herpes simplex virus. Methods for producingand purifying such vectors are know in the art. Preferably, the viralvector is a gamma-retrovirus or a lentivirus. The lentivirus may be amodified HIV virus suitable for use in delivering genes. The lentivirusmay be a SIV, FIV, or equine infectious anemia virus (EQIA) basedvector. The viral vector may comprise a targeting molecule to ensureefficient transduction with the nucleic acid sequence or nucleic acidconstruct. The targeting molecule will typically be provided wholly orpartly on the surface of the viral vector in order for the molecule tobe able to target the virus to T-cells. The viral vector is preferablyreplication deficient.

The vector may be a non-viral vector. Preferably, the non-viral vectoris a DNA plasmid, a naked nucleic acid, a nucleic acid complexed with adelivery vehicle, or an artificial virion. The non-viral vector may be ahuman artificial chromosome, as described in e.g. Kazuki et al., Mol.Ther. 19 (9): 1591-1601 (2011), and Kouprina et al., Expert Opinion onDrug Delivery 11 (4): 517-535 (2014). When the non-viral vector is anucleic acid complexed with a delivery vehicle, the delivery vehicle maybe a liposome, virosome, or immunoliposome. Integration of a plasmidvector may be facilitated by a transposase such as sleeping beauty orPiggyBAC.

T-Cells of the Invention

The invention provides a T-cell comprising (a) a nucleic acid sequenceof the invention or a nucleic acid construct of the invention, or (b) avector of the invention. Nucleic acid sequences, nucleic acid constructsand vectors are described in detail above.

The T-cell may be any type of T-cell. The T-cell may be a CD4+ T-cell,or helper T-cell (T_(H) cell), such as a T_(H)1, T_(H)2, T_(H)3,T_(H)17, T_(H)9, or T_(FH) cell. The T-cell may be a CD8+ T-cell, orcytotoxic T-cell. The T-cell may be a CD4+ or CD8+ memory T-cell, suchas a central memory T-cell or an effector memory T-cell. The T-cell maybe a regulatory T-cell (Treg).

The T-cell may comprise one or more, such as two or more, three or more,four or more, five or more or ten or more, nucleic acid sequencesencoding a CAR, independent of any CAR-encoding nucleic acid sequencecomprised in the construct or vector of the invention. When the T-cellcomprises two or more sequences encoding a CAR, the sequences may encodethe same CAR or different CARs. T-cell may therefore express one or moreCARs, independent of any CAR expressed from the construct or vector ofthe invention. Thus, the T-cell may be a CAR.

The T-cell may comprise one or more, such as two or more, three or more,four or more, five or more or ten or more nucleic acid sequences of theinvention. Similarly, the T-cell may comprise one or more, such as twoor more, three or more, four or more, five or more or ten or morenucleic acid constructs of the invention. The T-cell may comprise one ormore, such as two or more, three or more, four or more, five or more orten or more vectors of the invention. When the T-cell comprises two ormore nucleic acid sequences, nucleic acid constructs or vectors, the twoor more sequences, constructs or vectors may be the same or different.

Methods for producing the T-cell of the invention are set out below. TheT-cell is typically produced from a human T-cell. The T-cell of theinvention is therefore typically human. Alternatively, the T-cell may beproduced from a T-cell from another animal or mammal, for instance froma commercially farmed animal, such as a horse, cattle, a sheep or a pig,from a laboratory animal, such as a mouse or a rat, or from a petanimal, such as a cat, a dog, a rabbit or a guinea pig. The T-cell maytherefore be equine, bovine, ovine, porcine, murine, feline, leporine orcavine. The T-cell may be produced from a haematopoietic stem cell.Thus, the T-cell may be produced from cord blood. The T-cell may beproduced from an embryonic stem cell, or an induced pluripotent stemcell (iPS cell).

The T-cell may have reduced or completely eliminated expression ofnative TCR. Accordingly, the T-cell may have a reduced or completelyeliminated capacity to induce GVHD following administration to aHLA-mismatched recipient or patient.

Methods

The invention also provides a method for producing a T-cell expressing amolecule of the invention. The method comprises: transfecting ortransducing a T-cell with a nucleic acid sequence of the invention or anucleic acid construct of the invention; and expressing the molecule inthe T-cell.

The T-cell that is transfected or transduced is typically a humanT-cell. The human T-cell is typically from a donor that is HLA-matchedor HLA-mismatched compared to a patient into which the resultant T-cellwill be administered, or from a pool or bank of donor T-cells. TheT-cell may alternatively be from another animal or mammal, for instancefrom a commercially farmed animal (such as a horse, cattle, a sheep or apig), a laboratory animal (such as a mouse or a rat), or from a petanimal (such as a cat, a dog, a rabbit or a guinea pig). In thisinstance, when the resultant T-cell is administered to a patient, thepatient is typically the same type of animal or mammal as that fromwhich the initial T-cell is obtained.

The T-cell may be derived from a haematopoietic stem cell. Thus, theT-cell may be from cord blood. The T-cell may be derived from anembryonic stem cell, or an induced pluripotent stem cell (iPS cell).

The T-cell may be any type of T-cell that expresses a TCR comprising analpha-chain and a beta-chain. T-cell types are described in detail abovewith reference to the T-cell of the invention. Preferably, the T-cell isa CAR T-cell.

The T-cell is typically obtained from the donor using leucapheresis. TheT-cell may be sorted from the product of leucaphereis, for example if amixed population of leukocytes or lypmhocytes is obtained fromleucapheresis. T-cells may be isolated from the leucapheresis productusing any means known in the art. For instance, T-cells may be sortedfrom the product using FACS or magnetic-activated cell sorting (MACS).

The T-cell provided may be obtained from a sample taken from the donor.The sample is typically a blood sample. The sample may be a bone marrowsample, or a sample of a lymphoid tissue such as a lymph node, tonsil,spleen or thymus. If the sample is obtained from bone marrow or alymphoid tissue, the cells in the sample may need to be dissociated toallow T-cells to be isolated. Methods for dissociating bone marrow andlymphoid tissues are known in the art, but may include maceration (forexample, maceration through a cell strainer) or enzymatic digestion.T-cells may be isolated from the sample or dissociated sample using anymeans known in the art. For instance, T-cells may be sorted from thesample using FACS or magnetic-activated cell sorting (MACS).

T-cells obtained from the donor may be expanded prior to use in themethod of the invention. Expansion may involve stimulating the T-cellsusing an agonist for the TCR/CD3 complex, such as an anti-CD3 antibody,and/or an agonist for CD28 signalling, such as an anti-CD28 agonist. Theanti-CD3 antibody and/or the anti-CD28 antibody may be loaded to a beador other particle. Culture conditions for T-cell expansion are wellknown in the art.

Once provided, the T-cell is transfected or transduced with a nucleicacid sequence of the invention or a nucleic acid construct of theinvention. Nucleic acid sequences and nucleic acid constructs aredescribed in detail above.

The term “transduction” may be used to describe virus-mediated nucleicacid transfer. A viral vector may be used to transduce the cell with theone or more constructs. Conventional viral based expression systemscould include retroviral, lentivirus, adenoviral, adeno-associated (AAV)and herpes simplex virus (HSV) vectors for gene transfer. Methods forproducing and purifying such vectors are know in the art. The vector ispreferably a vector of the invention. The T-cell may be transduced usingany method known in the art. Transduction may be in vitro or ex vivo.

The term “transfection” may be used to describe non-virus-mediatednucleic acid transfer. The T-cell may be transfected using any methodknown in the art. Transfection may be in vitro or ex vivo. Any vectorcapable of transfecting the T-cell may be used, such as conventionalplasmid DNA or RNA transfection. A human artificial chromosome and/ornaked RNA and/or siRNA may be used to transfect the cell with thenucleic acid sequence or nucleic acid construct. Human artificialchromosomes are described in e.g. Kazuki et al., Mol. Ther. 19 (9):1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery11 (4): 517-535 (2014). Alternative non-viral delivery systems includeDNA plasmids, naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Methods of non-viral delivery ofnucleic acids include lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,4,946,787; and 4,897,355) and lipofection reagents are sold commercially(e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids thatare suitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Nanoparticle delivery systems may be used to transfect the cell with thenucleic acid sequence or nucleic acid construct. Such delivery systemsinclude, but are not limited to, lipid-based systems, liposomes,micelles, microvesicles and exosomes. With regard to nanoparticles thatcan deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci USA. 2013Aug. 6; 110 (32): 12881-6; Zhang et al., Adv Mater. 2013 Sep. 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13 (3): 1059-64;Karagiannis et al., ACS Nano. 2012 Oct. 23; 6 (10):8484-7; Whitehead etal., ACS Nano. 2012 Aug. 28; 6 (8):6922-9 and Lee et al., NatNanotechnol. 2012 Jun. 3; 7 (6):389-93. Lipid Nanoparticles, SphericalNucleic Acid (SNA(tm)) constructs, nanoplexes and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means fordelivery of a construct or vector in accordance with the invention.

Uptake of nucleic acid constructs may be enhanced by several knowntransfection techniques, for example those including the use oftransfection agents. Examples of these agents includes cationic agents,for example, calcium phosphate and DEAE-Dextran and lipofectants, forexample, lipofectAmine, fugene and transfectam.

The T-cell may be transfected under suitable conditions. The T-cell andagent or vector may, for example, be contacted for between five minutesand ten days, preferably from an hour to five days, more preferably fromfive hours to two days and even more preferably from twelve hours to oneday.

The nucleic acid sequence or nucleic acid construct transduced ortransfected into the T-cell gives rise to expression of the molecule ofthe invention in the T-cell. As set out above, the nucleic acidconstruct may comprise a sequence encoding a CAR and/or a sequenceencoding a suicide gene, in addition to the sequence encoding themolecule of the invention. Thus, the nucleic acid construct transducedor transfected into the T-cell may also give rise to expression of theCAR and/or the suicide gene. The nucleic acid sequence or nucleic acidconstruct preferably comprises a promoter which is operably linked tothe one or more of the encoded sequences, and which is active in theT-cell or which can be induced in the T-cell.

The invention further provides a method for reducing or completelyeliminating expression of native TCR in a T-cell, comprising (a)providing a nucleic acid sequence of the invention or a nucleic acidconstruct of the invention, or a vector encoding the nucleic acidsequence or nucleic acid construct; (b) transfecting or transducing theT-cell with the nucleic acid sequence, nucleic acid construct or vector;and (c) expressing the molecule in the T-cell. The method may beconducted in vivo. Preferably, the method is conducted in vitro.T-cells, nucleic acid sequences, nucleic acid constructs, vectors,transfection, transduction and expression are discussed in detail above.The T-cell is preferably a CAR T-cell. CAR T-cells are described indetail above. The CAR T-cell may be specific for one or more tumorantigens, for example one or more antigens associated with AML. The CART-cell may be specific for one or more of CD33, CD123 and CLL1. Theresulting T-cell may have a reduced or completely eliminated capacity tocause GVHD following administration to a HLA-mismatched recipient orpatient.

The invention also provides a method for producing a T-cell havingreduced or completely eliminated expression of native TCR, comprising(a) providing a T-cell; (b) transfecting or transducing the T-cell witha nucleic acid sequence of the invention or a nucleic acid construct ofthe invention, or a vector encoding the nucleic acid sequence or nucleicacid construct; and (c) expressing the molecule in the T-cell. T-cells,nucleic acid sequences, nucleic acid constructs, vectors, transfection,transduction and expression are discussed in detail above. The T-cellproduced by the method may be a CAR T-cell, as described in detailabove. In this case, the T-cell provided may be transfected ortransduced with a nucleic acid construct comprising a first nucleic acidsequence encoding the molecule of the invention, a second nucleic acidsequence encoding a CAR and, optionally, a third nucleic acid sequenceencoding a suicide gene, or a vector comprising the construct. Asmentioned above, the advantage of producing CAR T-cells having reducedor completely eliminated expression of native TCR in this way is thatCAR expression is linked to expression of the molecule that inhibitsnative TCR expression and, optionally, expression of the suicide gene.Sorting on the expression of one marker therefore also sorts forexpression of the other marker(s).

Medicaments, Methods and Therapeutic Use

The invention provides a method of reducing or preventing graft versushost disease (GVHD) in a patient associated with the administration ofone or more allogeneic CAR T-cells to the patient, comprising (a)transfecting or transducing the one or more CAR T-cells with a nucleicacid sequence of the invention or a nucleic acid construct of theinvention, or a vector encoding the nucleic acid sequence or nucleicacid construct; and (b) administering the CAR T-cells to the patient.The invention also provides a nucleic acid sequence of the invention ora nucleic acid construct comprising a first nucleic acid sequence of theinvention and a second nucleic acid sequence which encodes a suicidegene for use in a method of reducing or preventing GVHD in a patientassociated with the administration of one or more CAR T-cells to thepatient, the method comprising (a) transfecting or transducing the oneor more CAR T-cells with the nucleic acid sequence or nucleic acidconstruct and (b) administering the CAR T-cells to the patient. In theseaspects, the invention relates to reducing the ability of therapeuticCAR T-cells to cause GVHD in a patient.

The invention also provides a method of reducing or preventing GVHD in apatient associated with the transfusion of one or more CAR T-cells tothe patient, comprising (a) generating the one or more CAR T-cells bytransfecting or transducing one or more T-cells with a nucleic acidconstruct comprising a first nucleic acid sequence of the invention anda second nucleic acid sequence which encodes a chimeric antigenreceptor, or a vector encoding the nucleic acid construct; and (b)administering the CAR T-cells to the patient. In addition, the inventionprovides a nucleic acid construct comprising a first nucleic acidsequence of the invention and a second nucleic acid sequence whichencodes a chimeric antigen receptor for use in a method of reducing orpreventing GVHD in a patient associated with the administration of oneor more CAR T-cells to the patient, the method comprising (a) generatingthe one or more CAR T-cells by transfecting or transducing one or moreT-cells with the nucleic acid construct and (b) administering the CART-cells to the patient. In these aspects, the invention relates to theproduction of CAR T-cells having a reduced ability to cause GVHD in apatient.

The invention further provides a method of reducing or preventing GVHDin a patient associated with the transfusion of one or more CAR T-cellsto the patient, comprising (a) producing one or more T-cells expressinga molecule of the invention by (i) providing one or more T-cells; (ii)transfecting or transducing the one or more T-cells with a nucleic acidsequence of the invention or a nucleic acid construct comprising a firstnucleic acid sequence of the invention and a second nucleic acidsequence which encodes a suicide gene, or a vector encoding the nucleicacid sequence or nucleic acid construct; and (iii) expressing themolecule in the one or more T-cells; (b) converting the one or moreT-cells into one or more CAR T-cells by transfecting or transducing theone or more T-cells with a construct encoding a CAR and expressing theCAR; and (c) administering the CAR T-cells to the patient. Thus, in thisaspect, the invention relates to directing a T-cell having reducedability to cause GVHD in a patient towards an antigen of interest in thepatient.

GVHD may be reduced in one or more ways. Firstly, “reducing GVHD” mayrefer to reducing the incidence of GVHD in patients transfused oradministered with one or more CAR T-cells. For example, the incidence ofGVHD may be reduced in patients transfused or administered with one ormore CAR T-cells transfected or transduced with a nucleic acid sequenceor nucleic acid construct of the invention compared to patientstransfused or administered with one or more CAR T-cells that is neithertransfected nor transduced with a nucleic acid sequence or nucleic acidconstruct of the invention.

Secondly, “reducing GVHD” may refer to reducing the severity of GVHD inpatients transfused or administered with one or more CAR T-cells. Forinstance, the severity of GVHD may be reduced in patients transfused oradministered with one or more CAR T-cells transfected or transduced witha nucleic acid sequence or nucleic acid construct of the inventioncompared to patients transfused or administered with one or more CART-cells that is neither transfected nor transduced with a nucleic acidsequence or nucleic acid construct of the invention. In particular, thepatients may experience fewer clinical signs of GVHD, or less severeclinical signs of GVHD. The patients may experience a lower grade and/orstage of GVHD.

In these ways, the potential of therapeutic CAR T-cells to causeundesirable side effects in an HLA (or equivalent) -mismatched patientcan be minimised. The resultant CAR T-cell product may therefore be usedoff-the-shelf in any patient in need thereof, regardless of thepatient's HLA (or equivalent) type. Accordingly, the methods of theinvention permit the production of an improved and safer CAR T-cellproduct.

The patient may be any suitable patient. The patient is generally ahuman patient. The patient may be any of the animals or mammalsmentioned above with reference to the source of the T-cells.

The CAR may be any suitable CAR. Suitable CARs are described above withreference to the construct of the invention. In particular, the CAR maycomprise an antigen recognition domain that is specific for a tumourantigen, a viral antigen, a bacterial antigen, a fungal antigen, aprotozoal antigen, a host antigen or a cytokine. The antigen recognitiondomain is preferably specific for a tumour antigen, such as an antigenassociated with AML. The antigen recognition domain may be specific foran antigen associated with any of the diseases discussed below.

Nucleic acid sequences and constructs, vectors, and transfection,transduction and expression are discussed in detail above.

The invention provides a method of treating a disease in a patient inneed thereof, comprising administering to the patient a therapeuticallyeffective number of CAR T-cells expressing a molecule of the invention.Similarly, the invention provides CAR T-cells expressing a molecule ofthe invention, for use in a method of treating a disease in a patient inneed thereof, the method comprising administering to the patient atherapeutically effective number of cells.

The disease may be any disease in which the patients may benefit fromantigen-specific T-cell responses. For instance, the disease may be adisease in which the subject may benefit from increased cytotoxic,helper or gamma delta T-cell responses. The disease may be an infection,such as a bacterial, viral, fungal, protozoal or other parasiticinfection. Preferably, the disease is cancer. The cancer may be analcancer, bile duct cancer (cholangiocarcinoma), bladder cancer, bloodcancer, bone cancer, bowel cancer, brain tumours, breast cancer,colorectal cancer, cervical cancer, endocrine tumours, eye cancer (suchas ocular melanoma), fallopian tube cancer, gall bladder cancer, headand/or neck cancer, Kaposi's sarcoma, kidney cancer, larynx cancer,leukaemia, liver cancer, lung cancer, lymph node cancer, lymphoma,melanoma, mesothelioma, myeloma, neuroendocrine tumours, ovarian cancer,oesophageal cancer, pancreatic cancer, penis cancer, primary peritonealcancer, prostate cancer, Pseudomyxoma peritonei, skin cancer, smallbowel cancer, soft tissue sarcoma, spinal cord tumours, stomach cancer,testicular cancer, thymus cancer, thyroid cancer, trachea cancer,unknown primary cancer, vagina cancer, vulva cancer or endometrialcancer. The leukaemia is preferably acute lymphoblastic leukaemia, acutemyeloid leukaemia (AML), chronic lymphocytic leukaemia or chronicmyeloid leukaemia. The lymphoma may be Hodgkin lymphoma or non-Hodgkinlymphoma. The cancer may be primary cancer or secondary cancer.Preferably, the cancer is leukaemia. Most preferably, the cancer is AML.When the cancer is AML, the one or more CAR T-cells are preferablyspecific for one or more of CD33, CD 123 and CLL1.

When the cancer is AML, it may be advantageous for the expression ofnative TCR in the CAR T-cells to be reduced rather than completelyeliminated. By allowing some native TCR expression to remain,administration of the CAR T-cells to the patient may be associated witha small amount of GVHD. This weak GVHD may be of benefit to the patient,as it may non-specifically attack the bone marrow compartment, killingAML cells and their precursors. Non-specific attack of the marrowcompartment may necessitate the provision of a rescue allograft to thepatient. However, as administration of CAR T-cells targeting one or moreof CD33, CD 123 and CLL1 may cause myeoloablation and necessitate rescueallograft in any case, the residual retention of native TCR expressionin such CAR T-cells does not have any particular disadvantages.

The disease may be a disease in which the subject may benefit fromincreased regulatory T-cell responses to an antigen. The disease may bean allergic disease, such as atopic dermatitis, allergic airwayinflammation or perennial allergic rhinitis. The disease is preferablyan autoimmune condition, such as alopecia areata, autoimmuneencephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis,dermatomyositis, diabetes (type 1), autoimmune juvenile idiopathicarthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome,idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmunemyocarditis, multiple sclerosis, pemphigus/pemphigoid, perniciousanemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis,psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis,Sjögren's syndrome, systemic lupus erythematosus, autoimmunethyroiditis, uveitis or vitiligo. The disease is preferably GVHD.

The T-cells or CAR T-cells of the invention may be provided as apharmaceutical composition. The pharmaceutical composition preferablycomprises a pharmaceutically acceptable carrier or diluent. Thepharmaceutical composition may be formulated using any suitable method.Formulation of cells with standard pharmaceutically acceptable carriersand/or excipients may be carried out using routine methods in thepharmaceutical art. The exact nature of a formulation will depend uponseveral factors including the cells to be administered and the desiredroute of administration. Suitable types of formulation are fullydescribed in Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company, Eastern Pa., USA.

The T-cells, CAR T-cells or pharmaceutical composition may beadministered by any route. Suitable routes include, but are not limitedto, intravenous, intramuscular, intraperitoneal or other appropriateadministration routes. The T-cells, CAR T-cells or pharmaceuticalcomposition is preferably administered intravenously.

Compositions may be prepared together with a physiologically acceptablecarrier or diluent. Typically, such compositions are prepared as liquidsuspensions of cells. The cells may be mixed with 25 an excipient whichis pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, of the like and combinations thereof.

In addition, if desired, the pharmaceutical compositions of theinvention may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance effectiveness. The composition preferably comprises humanserum albumin. 30

One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile,nonpyrogenic isotonic solution for intravenous administration. Each 100mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of SodiumGluconate (C6H11NaO7); 368 mg of Sodium Acetate Trihydrate, USP(C2H3NaO2.3H2O); 37 mg of Potassium Chloride, USP (KCl); and 30 mg ofMagnesium Chloride, USP (MgCl2*6H20). It contains no antimicrobialagents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to8.0).

The T-cells or CAR T-cells are administered in a manner compatible withthe dosage formulation and in such amount will be therapeuticallyeffective. The quantity to be administered depends on the subject to betreated, the disease to be treated, and the capacity of the subject'simmune system. Precise amounts of T-cells or CAR T-cells required to beadministered may depend on the judgement of the practitioner and may bepeculiar to each subject.

Any suitable number of T-cells or CAR T-cells may be administered to asubject. For example, at least, or about, 0.2×10⁶, 0.25×10⁶, 0.5×10⁶,1.5×10⁶, 4.0×10⁶ or 5.0×10⁶ cells per kg of patient may administered.For example, at least, or about, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ cells may beadministered. As a guide, the number of cells of the invention to beadministered may be from 10⁵ to 10⁹, preferably from 10⁶ to 10⁸.Typically, up to 2×10⁸ IMP cells are administered to each patient. Insuch cases where cells are administered or present, culture medium maybe present to facilitate the survival of the cells. In some cases thecells of the invention may be provided in frozen aliquots and substancessuch as DMSO may be present to facilitate survival during freezing. Suchfrozen cells will typically be thawed and then placed in a buffer ormedium either for maintenance or for administration.

The following Examples illustrate the invention.

EXAMPLES Example 1 Knockdown of the TCR/CD3 Complex

ScFvs were cloned into a retroviral transfer vector in frame at theiramino-terminus with a signal peptide and with the SEKDEL (SEQ ID NO:20)motif at their carboxy terminus. An internal ribosomal entry sequence(IRES) was cloned after the scFv-SEKDEL. A fluorescent protein wascloned after the IRES. 293T cells were transfected with the transfervectors along with an expression plasmid coding for the RD114 envelopeand a further expression plasmid which supplies gamma-retroviral gagpol.After 48 and 72 hours, supernatant was harvested. T-cell lines weretransduced by exposing the T-cells to the supernatant in the presence ofretronectin. Primary human T-cells were transduced in a similar mannerbut were stimulated with anti-CD3 and anti-CD28 and IL2 beforehand. Todetermine TCR surface expression, T-cells were stained with fluorescentmonoclonal antibodies which recognize CD3/TCR, washed and analysed byflow-cytometry.

In initial experiments, the scFvs from hybridomas OKT3 and BMA031 wereused. Some reduction in TCR expression was noted (FIG. 1). In addition,TCR knockdown in non-transduced T-cells in the same culture system wereobserved. This suggested that the scFv was leaking out of the cells andblocking staining for the TCR rather than blocking its maturation andexit to the cell surface.

Consequently, similar constructs were generated from other anti-TCR/CD3hybridomas. These were carefully selected from the literature on thebasis that they are known to be able to stain intracellularly—hencebeing able to recognize the assembling TCR/CD3 complex in a nascentstage before access to the Golgi has passed. The anti-TCR/CD3 antibodyUCHT1 (Beverley, P. C. & Callard, R. E. Distinctive functionalcharacteristics of human ‘T’ lymphocytes defined by E resetting or amonoclonal anti-T cell antibody. Eur. J. Immunol. 11, 329-334 (1981))was chosen for this application. FIG. 2 shows the amino acid sequence ofthe S-UCHT1-KDEL molecule. FIG. 3a depicts the structure of thismolecule, and FIG. 3b shows its presumed mechanism of action.

Jurkat T-cells were transduced with this construct and stained withanti-TCR/CD3 antibodies. The retention protein was co-expressed with thefluorescent marker protein eBFP2. The clone of staining antibody wascarefully chosen as to not compete with UCHT1 binding. As a control,Jurkat T-cells which have had their TCR alpha and beta chain genesdisrupted were also stained. Complete reduction of surface TCR wasobserved in transduced (but not non-transduced Jurkat T-cells) (FIGS.4aand 4b ).

Peripheral blood T-cells from normal donors were transduced withUCHT1-sekdel. Transduced T-cells were stained with a non-cross-reactiveanti-CD3/TCR mAb and analysed by flow-cytometry. Lack of TCR expressionwas seen on T-cells expressing the marker gene (FIG. 5). Fluorescentsignal was down to that of isotype control in this population.

SEQUENCE LISTING CDR1 of UCHT1 (Humanised) VH SEQ ID NO: 1 GYSFTGYTCDR2 of UCHT1 (Humanised) VH SEQ ID NO: 2 INPYKGVSCDR3 of UCHT1 (Humanised) VH SEQ ID NO: 3 ARSGYYGDSDWYFDVCDR1 of UCHT1 (Humanised) VL SEQ ID NO: 4 QDIRNYCDR2 of UCHT1 (Humanised) VL SEQ ID NO: 5 YTSCDR3 of UCHT1 (Humanised) VL SEQ ID NO: 6 QQGNTLPWTHCVR of UCHT1 (Humanised) SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSLCVR of UCHT1 (Humanised) SEQ ID NO: 8DIQMTQSPSSLSASVGNRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKVEIKCDR1 of Jovi.1 (Murine) VH SEQ ID NO: 9 GYTFTGYCDR2 of Jovi.1 (Murine) VH SEQ ID NO: 10 NPYNDDCDR3 of Jovi.1 (Murine) VH SEQ ID NO: 11 GAGYNFDGAYRFFDFCDR1 of Jovi.1 (Murine) VL SEQ ID NO: 12 RSSQRLVHSNGNTYLHCDR2 of Jovi.1 (Murine) VL SEQ ID NO: 13 RVSNRFPCDR3 of Jovi.1 (Murine) VL SEQ ID NO: 14 SQSTHVPYTHCVR of Jovi.1 (Murine) SEQ ID NO: 15EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDFWGQGTTLTVS LCVR of Jovi.1 (Murine) SEQ ID NO: 16DVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVP YTFGGGTKLEIKRKDEL target peptide sequence SEQ ID NO: 17 KDEL Adenoviral E19 proteinSEQ ID NO: 18 KYKSRRSFIDEKKMP HLA Invariant Chain SEQ ID NO: 19MHRRRSRSCR SEKDEL retention sequence SEQ ID NO: 20 SEKDEL S-UCHT1-KDELSEQ ID NO: 21 METDTLLLWVLLLWVPGDIQMTQSPSSLSASVGNRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSGGGGSGGGGSGGGGSVLEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTILINKKDELSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSDPAEPSEKDEL UCHT1(Humanised) VL SEQ ID NO: 22DIQMTQSPSSLSASVGNRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKVEIKUCHT1 (Humanised) VH SEQ ID NO: 23EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSJOVI-1(Murine) VL SEQ ID NO: 24DVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVP YTFGGGTKLEIKRJOVI-1(Murine) VH SEQ ID NO: 25EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDFWGQGTTLTVS

1. A molecule which disrupts the expression of native T cell receptor(TCR) in a T cell, which molecule comprises a binding portion comprisinga binding domain which binds to one or more components of the TCR/CD3complex, and a retention portion comprising a retention domain thatretains the one or more components within the endoplasmic reticulum (ER)or Golgi apparatus.
 2. A molecule according to claim 1, wherein the oneor more components are not assembled to form the TCR/CD3 complex.
 3. Amolecule according to claim 1, wherein the one or more components areassembled to form the TCR/CD3 complex.
 4. A molecule according claim 1,wherein the binding domain is a single chain variable fragment (scFv), ascFv-Fc, a single-domain antibody, or a monoclonal antibody.
 5. Amolecule according to claim 4, wherein the single-domain antibody is acamelid antibody, an artificial V_(H)H fragment or an IgNAR.
 6. Amolecule according to claim 1, wherein the binding domain binds toCD3-epsilon (CD3ε) or TCR beta chain.
 7. A molecule according to claim6, wherein the binding domain is a UCHT1 antibody or a Jovi.1 antibody,or is derived from a UCHT1 antibody or a Jovi.1 antibody.
 8. A moleculeaccording to claim 6, wherein the binding domain comprises (a) threeheavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within the HCVRsequence of SEQ ID NO: 7 and/or three light chain CDRs (LCDR1, LCDR2 andLCDR3) contained within the LCVR sequence of SEQ ID NO: 8, or (b) threeheavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within the HCVRsequence of SEQ ID NO: 15 and/or three light chain CDRs (LCDR1, LCDR2and LCDR3) contained within the LCVR sequence of SEQ ID NO:
 16. 9. Amolecule according to claim 1, wherein the retention domain is KDELsequence (SEQ ID NO: 17), a KKXX motif (SEP ID NO:26), a KXKXX motif(SEQ ID NO:27), a tail of adenoviral E19 protein having sequenceKYKSRRSFIDEKKMP (SEQ ID NO: 18), or a fragment of HLA invariant chainhaving sequence MHRRRSRSCR (SEQ ID NO:19).
 10. A molecule according toclaim 1, wherein the retention domain is a KDEL sequence.
 11. A moleculeaccording to claim 1, wherein the retention domain is C-terminal to thebinding domain.
 12. A nucleic acid sequence encoding a molecule asdefined in claim 1, comprising a nucleic acid sequence encoding abinding portion comprising a binding domain which binds to one or morecomponents of TCR/CD3 complex, and a retention portion comprising aretention domain that retains the one or more components within the ERor Golgi apparatus.
 13. A nucleic acid construct comprising (a) a firstnucleic acid sequence, a second nucleic acid sequence which encodes achimeric antigen receptor (CAR) and optionally a third nucleic acidsequence which encodes a suicide gene, or (b) a first nucleic acidsequence and a second nucleic acid binding portion comprising a bindingdomain which binds to one or more components of TCR/CD3 complex, and aretention portion comprising a retention domain that retains the one ormore components within the ER or Golgi apparatus.
 14. (canceled) 15.(canceled)
 16. A nucleic acid construct according to claim 13, wherein(a) the suicide gene is RQR8, iCasp9 or thymidine kinase and/or (b) oneor more of the nucleic acid sequences are linked by a nucleic acidsequence encoding a foot-and-mouth disease 2A-like peptide. 17.(canceled)
 18. A vector comprising the nucleic acid sequence accordingto claim
 12. 19. A vector according to claim 18, wherein the vector is aviral vector or a non-viral vector.
 20. A vector according to claim 19,wherein the viral vector is a retrovirus, a gamma-retrovirus, alentivirus, an adenovirus, an adeno-associated virus, a vaccinia virusor a herpes simplex virus.
 21. (canceled)
 22. (canceled)
 23. A vectoraccording to claim 19, wherein the non-viral vector is a DNA plasmid, anaked nucleic acid, a nucleic acid complexed with a delivery vehicle, oran artificial virion.
 24. A vector according to claim 23, wherein thedelivery vehicle is a liposome, virosome, or immunoliposome.
 25. Amethod for producing a T cell expressing a molecule that disrupts theexpression of native T cell receptor (TCR) in a T cell and thatcomprises a binding portion comprising a binding domain which binds toone or more components of the TCR/CD3 complex, and that comprises aretention portion comprising a retention domain that retains the one ormore components within the endoplasmic reticulum (ER) or Golgiapparatus, the method comprising: (a) transfecting or transducing a Tcell with a nucleic acid sequence that encodes the molecule, ortransfecting or transducing a T cell with a nucleic acid construct asdefined in claim 13; and (b) expressing the molecule in the T cell. 26.A method according to claim 25, wherein the transfecting or transducingof step (a) is performed using a vector that comprises nucleic acidsequence that encodes a binding portion comprising a binding domainwhich binds to one or more components of TCR/CD3 complex, and aretention portion comprising a retention domain that retains the one ormore components within the ER or Golgi apparatus.
 27. A T cellcomprising (a) a nucleic acid sequence encoding a binding portioncomprising a binding domain which binds to one or more components ofTCR/CD3 complex, and a retention portion comprising a retention domainthat retains the one or more components within the ER or Golgi apparatusor a nucleic acid construct as defined in claim 13, or (b) a vector thatcomprises nucleic acid sequence that encodes a binding portioncomprising a binding domain which binds to one or more components ofTCR/CD3 complex, and a retention portion comprising a retention domainthat retains the one or more components within the ER or Golgiapparatus.
 28. A T cell according to claim 27, wherein (a) the nucleicacid sequence encoding the molecule is incorporated to the genome of thecell and/or (b) the nucleic acid construct comprises a nucleic acidsequence encoding a CAR and/or a nucleic acid sequence encoding asuicide gene, and wherein the nucleic acid sequence encoding the CARand/or the nucleic acid sequence encoding a suicide gene is incorporatedto the genome of the cell.
 29. (canceled)
 30. A method for reducing orcompletely eliminating expression of native TCR in a T cell, comprising(a) providing a nucleic acid sequence encoding a binding portioncomprising a binding domain which binds to one or more components ofTCR/CD3 complex, and a retention portion comprising a retention domainthat retains the one or more components within the ER or Golgi apparatusor a nucleic acid construct as defined in claim 13, or a vector encodingthe nucleic acid sequence or nucleic acid construct; (b) transfecting ortransducing the T cell with the nucleic acid sequence, nucleic acidconstruct or vector; and (c) expressing the molecule in the T cell. 31.A method for producing a T cell having reduced or completely eliminatedexpression of native TCR, comprising (a) providing a T cell; (b)transfecting or transducing the T cell with a nucleic acid sequenceencoding a binding portion comprising a binding domain which binds toone or more components of TCR/CD3 complex, and a retention portioncomprising a retention domain that retains the one or more componentswithin the ER or Golgi apparatus or a nucleic acid construct as definedin claim 13, or a vector encoding the nucleic acid sequence or nucleicacid construct; and (c) expressing the molecule in the T cell.
 32. Amethod of reducing or preventing graft versus host disease (GVHD) in apatient associated with the administration of one or more CAR T cells tothe patient, comprising: (I) (a) transfecting or transducing the one ormore CAR T cells with a nucleic acid sequence encoding a binding portioncomprising a binding domain which binds to one or more components ofTCR/CD3 complex, and a retention portion comprising a retention domainthat retains the one or more components within the ER or Golgi apparatusor a nucleic acid construct as defined in claim 13, or a vector encodingthe nucleic acid sequence or nucleic acid construct; and (b)administering the CAR T cells to the patient; (II) (a) generating theone or more CAR T cells by transfecting or transducing one or more Tcells with a nucleic acid construct as defined in claim 13, or a vectorencoding the nucleic acid construct; and (b) administering the CAR Tcells to the patient; or (III) (a) producing one or more T cellsexpressing a molecule as defined in claim 1 by (i) providing one or moreT cells; (ii) transfecting or transducing the one or more T cells with anucleic acid sequence encoding a binding portion comprising a bindingdomain which binds to one or more components of TCR/CD3 complex, and aretention portion comprising a retention domain that retains the one ormore components within the ER or Golgi apparatus or a nucleic acidconstruct as defined in claim 13, or a vector encoding the nucleic acidsequence or nucleic acid construct; and (iii) expressing the molecule inthe one or more T cells; (b) converting the one or more T cells into oneor more CAR T cells by transfecting or transducing the one or more Tcells with a construct encoding a CAR and expressing the CAR; and (c)administering the CAR T cells to the patient.
 33. (canceled) 34.(canceled)
 35. A method according to claim 32, wherein the patient ishuman.
 36. A method according to claim 32, wherein the CAR comprises anantigen recognition domain that is specific for a tumour antigen, aviral antigen, a bacterial antigen, a fungal antigen, a protozoalantigen, a host antigen, or a cytokine.
 37. (canceled)
 38. (canceled)39. (canceled)
 40. (canceled)
 41. A method of treating a disease in apatient in need thereof, comprising administering to the patient atherapeutically effective number of CAR T cells expressing a molecule asdefined in claim
 1. 42. A method according to claim 41, wherein thedisease is cancer.
 43. A method according to claim 42, wherein thecancer is acute myeloid leukaemia.
 44. A method according to claim 43,wherein one or more of the CAR T cells are specific for one or more ofCD33, CD 123 and CLL1.
 45. A method according to claim 41, wherein thedisease is an autoimmune condition.
 46. (canceled)